The long-term objective of this application is to understand the mechanisms regulating calcium metabolism and the role of calcium in tissue morphogenesis and physiological functions during embryonic development. Chick embryos will be used as the experimental model and the experiments will address the following specific aims: 1) to analyze the cellular and molecular mechanisms of calcium transport by extraembryonic membranes, i.e. the chorioallantoic membrane (CAM) and yolk sac (YS), using both cellular and cell-free approaches, with particular attention to the developmental and metabolic regulation of on of two calcium-binding proteins, transcalcin of the CAM and calbindin-D28K of the YS; 2) to elucidate the functional role of calcium and the mineralized matrix on cell differentiation in intramembranous bone development, in particular the possible mediating role of transforming growth factor beta; and 3) to determine the cellular pathways, In particular cellular calcium handling, involved in abnormal cardiovascular functions induced by systemic calcium deficiency during development. Chick embryos are uniquely suited for these studies since by long-term culture without the eggshell, their primary calcium source, they may be rendered highly calcium deficient. By comparing normal and shell-less (SL) embryos with respect to the above parameters, significant insights may be gained into the functional importance of calcium in embryogenesis. In summary, the studies proposed here should provide information on how Ca is translocated into the developing embryo to meet its needs, how systemic Ca homeostasis is regulated, and how its proper maintenance is important to skeletal development and cardiovascular functions. This multi-targeted project is realistically based on the overall research interests and demonstrated strength of the laboratory, and is expected to yield information useful and relevant to the general areas of fetal development, and skeletal and cardiovascular biology.